Superoxide anion is a short-lived oxygen radical released to the outside of appropriately stimulated leukocytes (monocytes, macrophages, and polymorphonuclear leukocytes). Synthesis involves a cytochrome b 558-containing plasma membrane enzyme complex called NADPH oxidase which transfers an electron to molecular oxygen, thus radicalizing it. The short-lived superoxide anion is a potent oxidant and can damage infectious organisms not protected by a countering superoxide-dismutation enzyme or scavenger. The dismutation product, hydrogen peroxide, is also toxic to a range of organisms.
As a front line immunologic response to infections, the importance of oxygen radical release to immunity is demonstrated in people who suffer from chronic granulomatous disease (CGD). These people lack NADPH oxidase activity. Without medical intervention, they suffer life threatening crises from infections that are normally controlled through this immune response. CGD is a disease caused by known genetic defects in components of the oxidase-excitation pathway. People with CGD have normal cell-mediated and humoral immunity; only their oxidase activity is defective. Theirs is a form of immunologic deficiency that is so specific in its cause that it clearly identifies the contribution of oxygen radicals to innate disease control.
The study of cellular pathways that activate the NADPH oxidase, and the components that comprise it, have received considerable attention in recent years. Ongoing projects are in the interests of basic science as well as of clinical research. At present, the methods most often used for detecting superoxide include measurements of: (1) the reduction of exogenously supplied cytochrome c; (2) uptake of oxygen from the medium (measured by the Clark electrode); and, (3) luminol-mediated chemiluminescence. The first method has limited sensitivity, and is complicated by the reoxidation of superoxide-reduced cytochrome c by contaminants and cell lysis. The oxygen uptake method is less sensitive than cytochrome c reduction and has the serious disadvantage of requiring prohibitively large numbers of cells per assay. The third method, luminol-mediated chemiluminescence, is more sensitive than the other two, with accurate readings extending over a range of signal spanning three orders of magnitude.
Luminol chemiluminescence assay measurement involves the measurement of superoxide anion produced by cell biochemistry. For the purposes of the present invention, luminol will also include a related variation known as isoluminol. Discussion of luminol is intended to include isoluminol. The chain of events is believed to involve cell surface receptors, such as those binding complexed immunoglobulin G (Fc receptors) or bacterial formylated peptides (f-met-leu-phe receptors), which are stimulated by their specific agonists. Any means for signal transduction to activate NADPH oxidase is usable with the assay of the present invention. Such means can be physiologic or mimetic of the physiologic pathway.
The activated receptor triggers the production or activation of NADPH oxidase, which binds to and radicalizes molecular oxygen by adding an electron. The radicalized oxygen, called superoxide anion, is subsequently released into the extracellular environment, where it can interact with luminol. Superoxide anion by itself oxidizes available lipid and proteins some of which chemiluminesce as a result. This type of chemiluminescence produces a very poor, inefficient means of measuring superoxide anion. However, the addition of luminol results in an amplified signal with a proportionality to superoxide produced by the cells.
Luminol chemiluminescence assay functions as a detection and measurement technique because of the ability of superoxide anion to cleave luminol. This cleavage results in the release of photons (light), which can be measured by a luminometer, or other appropriate instrument. It is estimated that the photon to superoxide ratio is 1:1000.
It would be advantageous to enhance the sensitivity of the chemiluminescence assay in order to more accurately detect or measure lower concentrations of superoxide anion than currently possible. Enhancement of the photon signal in an assay with a concentration of superoxide anion present would be desirable.
U.S. Pat. Nos. 4,949,182, issued Sep. 25, 1990, and 4,962,192, issued Oct. 9, 1990, both to Schaap, disclose light producing compounds and compositions providing enhanced chemiluminescence. The compounds disclosed are 1,2-dioxetanes of various structures, which can be triggered to produce light at room temperatures when cleaved by an activating agent. U.S. Pat. No. 4,835,101, issued to Kao et al., discloses a reagent for luminescence-monitored enzyme assays containing an enhancer of 4-iodophenol or 4-phenylphenol. U.S. Pat. No. 4,598,044, issued to Whitehead et al., discloses a phenolic derivative as an enhancer. U.S. Pat. No. 4,842,997, issued to Carter et al., discloses the use of 6-hydroxybenzothiazole.
Kits containing luminol-enhancement reagents for performing chemiluminescence assays are commercially available from companies such as U.S. Biochemicals, Amersham and BioRad as non-isotopic detection systems for Western and DNA blotting. Some enhancement reagents use phenolic-based compounds, such as iodophenol. However, such enhancers are toxic to live cells and denaturing to some components of subcellular systems and cannot be used for in situ assays. It would be desirable to have an enhancement reagent that is non-cytotoxic and that does not denature components for use in assaying the activity of live cells.